Project 1 - Ischemic Tolerance and Endothelial Protection Cerebral microvascular inflammation is a well-recognized secondary injury mechanism in focal stroke. Our preliminary studies demonstrate activation of endogenous anti-inflammatory mechanisms at the level of the cerebrovascular endothelium during periods of preconditioning-induced ischemic tolerance, which we hypothesize reduces postischemic neurovascular unit dysfunction. Our overall goal is to characterize this ischemia-tolerant endothelial phenotype and to elucidate its underlying mechanisms, using our new in vivo model of sustained (onemonth) long-term tolerance (LTT), as well as an in vitro model of cerebral endothelial cell tolerance. Specific Aim 1: Measure postischemic changes in endothelial-neutrophil adherence and infiltration, and endothelial expression of adhesion molecules and their transcriptional regulation by NFkB. Hypothesis: Tolerance results, in part, from reductions in ischemia-induced, NFkB-mediated endothelial adhesion molecule expression which prevents neutrophil adherence and infiltration. Specific Aim 2: Evaluate the spatial-temporal manifestations of improved blood-brain barrier (BBB) integrity in ischemic tolerance, focusing on endothelial tight junction proteins and basal lamina constituents, and the participation of neutrophil-derived proteases. Hypothesis: Stabilization of postischemic endothelial barrier function in ischemic tolerance results from the maintenance of both BBB tight junction and basal lamina integrity, in conjunction with a reduction in the release ofMMP-9 and elastase from neutrophils. Specific Aim 3: Determine if the production of specific neutrophil chemoattractant cytokines and chemokines, and the respective signaling and receptor pathways by which they promote endothelial- neutrophil adherence and BBB breakdown, are reduced in ischemic tolerance. Hypothesis: Postischemic reductions in proinflammatory cytokine/chemokine signaling (lead candidates: IL-lfi and MIP2) contribute to reductions in the magnitude of the cerebrovascular inflammatory endpoints investigated in Aims 1and2. The endogenous mechanisms for endothelial protection from ischemia-induced inflammation identified in this project may provide therapeutic targets for reducing neurovascular unit dysfunction in focal stroke. RELEVANCE TO PUBLIC HEALTH: Ischemic stroke triggers a microvascular inflammatory response that exacerbates brain injury. In animals, overall brain injury is reduced following hypoxic "preconditioning". We will document protection at the microcirculation level, and elucidate the mechanisms that specifically reduce microvascular inflammation following preconditioning. This will help in the identification of new vascular therapeutic targets for stroke.